Target identification is a vital step in the drug discovery process and represents a substantial hurdle to further development when large numbers of hit compounds are identified by high throughput phenotypic screening. This problem is especially challenging in the case of antimalarial drug discovery because of the prevalence of unconventional targets such as hemozoin, the formation of which is thought to be inhibited by nearly half the clinical antimalarials and many experimental compounds. A key feature of the success of these drugs is that hemozoin is derived from host hemoglobin and is therefore not mutable, thereby reducing the ability of P. falciparum to acquire resistance. Prior studies have shown that target identification is complicated by the fact that the ability to inhibit abiotic synthetic hemozoin (?-hematin) formation is a necessary, but not sufficient predictor of hemozoin inhibition in Plasmodium falciparum malaria parasites and conversely, decreased hemozoin formation in the parasite is not itself confirmation of direct inhibition of hemozoin formation. We hypothesize that direct measurement of increased unsequestered heme together with decreased hemozoin in the intra-erythrocytic parasite is the most consistent method of identifying hemozoin inhibitors and that the latter cause characteristic perturbations of the heme detoxification pathway that can be exploited in target deconvolution. We further hypothesize that these inhibitors occupy a distinct region of chemical space that can be mapped in silico. To achieve our objectives, the following specific aims are proposed: 1) Develop generalizable methods to measure and detect hemozoin inhibition in Plasmodium falciparum; 2) Use in silico methods to map hemozoin inhibition in chemical space; and 3) Develop a model of the full heme detoxification pathway. To realize these aims, the research will be conducted as a collaborative and synergistic project between Timothy Egan at the University of Cape Town (UCT), Katherine de Villiers at Stellenbosch University (SU), South Africa and David Fidock at the Columbia University Medical Center (CUMC), New York, NY. Generalizable analytical methods for measuring unsequestered heme will be developed at UCT and transferred to CUMC for investigation of compound collections available at that site. A laboratory strain expected to exhibit universally reduced susceptibility to hemozoin inhibitors will be generated at CUMC. In silico methods for mapping ?-hematin inhibitors will be developed at SU and screening via molecular docking performed at UCT. Validation of these methods will take place at UCT and SU. The input data for modeling the heme detoxification pathway will be collected at UCT, while the mathematical model of this pathway will be developed at SU. Validation of the model will be conducted at UCT and CUMC. We expect that this work will transform our ability to discern the role of hemoglobin degradation and hemozoin synthesis in the mode of action of antimalarials, and provide vital tools to identify novel inhibitors that are refractory to a rapid gain of resistance and can treat multidrug-resistant P. falciparum malaria.